Summary / Abstract Opioid abuse is now a full-fledged epidemic in the United States. The CDC estimates that over 165,000 people have died from prescription opioid overdoses since 1999, and that 3,900 people start nonmedical use of prescription opioids each day. As a result, opioid use disorder (OUD) costs society an estimated $55 billion per year?twice the annual budget of the entire NIH. Most individuals with OUD began taking opioids on the advice of their physician to manage pain from work injury or injury sustained during military service, often without any knowledge of the risks of dependency. It is increasingly clear that the risks of dependency and addiction were greatly underestimated by the scientific community as well, that the most commonly administered opioids have among the highest physical and physiological dependence potentials of any abused class of drugs. However, genetic differences within the nave population predispose certain individuals to OUD once they are exposed to opioids in a clinical setting or otherwise. These genetic differences result in mu-opioid receptor (MOR) phenotypes that differ in binding, desensitization and internalization behavior?differences which are associated with higher risk of addiction and higher severity in withdrawal symptoms (the biggest predictor of relapse). Paired agent imaging (PAI) is an established method that can quantify receptor-ligand binding potential (BP) in vivo, and could the ability to measure rate of receptor internalization if fluorescent agonist and antagonist pairs are used instead of untargeted/targeted agent pairs. These two parameters?internalization rate and binding potential?are hypothesized to predict risk of OUD in nave users and risk of relapse in individuals recovering from OUD. We hypothesize that these clinically important differences in opioid receptor expression and behavior can be measured by otoendoscopic paired agent imaging (OPAI) of the inner ear. Therefore, we seek R21 funding under the NIBIB ?trailblazer? opportunity to: (1) design and assemble a rigid otoendoscope to perform PAI of opioid binding kinetics in the inner ear, and (2) use otoendoscopic paired agent imaging (OPAI) to quantify receptor behavior during chronic opioid exposure and following naloxone-induced withdrawal using fluorescently-labeled opioid peptide agonist/antagonist pairs. The otoendoscope will consist of commercially available Storz endoscope (<2.5 mm diameter) coupled to a small module that splits the image into three bands (RGB, 700- and 800- fluorescence) and then transmits to a single sCMOS camera. A multi-LED light source of specific illumination and excitation bands is transmitted down the endoscope via the Storz light guide coupler. By the end of the project, we will address three hypotheses: (1) spiral ganglia cells of the inner ear can be imaged using an otoendoscope in order to quantify binding potential and internalization, (2) binding and internalization rate predict chronic opioid exposure and naloxone-induced withdrawal in a chinchilla model of OUD. The potential impact of this research is substantial: the ability to quantify the individual variations in opioid receptor behavior non-invasively could improve the diversion of individuals at-risk for OUD away from opioids, could prevent opioid abuse in patients using opioids for chronic pain management, and could aid in the recovery of OUD by stratifying severity of withdrawal and, by extension, relapse risk to provide individualized, appropriate support.